(i) Field of the Invention
The present invention relates to a nucleic sequence specific for the Mycobacterium avium-intracellulare complex, as well as particular fragments of this sequence, which are capable of acting as nucleic probes for the detection of a nucleic acid specific for strains belonging to the Mycobacterium avium-intracellulare complex or as nucleic primers in the amplification of DNA or RNA obtained from strains of the Mycobacterium avium-intracellulare complex in a biological sample.
The invention also relates to a method of detection of strains belonging to the Mycobacterium avium-intracellulare complex in a biological sample using nucleic acid fragments, as well as a kit for implementing this method.
(ii) Description of Related Art
The number of infections caused by opportunistic mycobacteria is increasing, partly because they are frequent in AIDS patients. According to bacteriological studies carried out over the past few years, the most important opportunistic bacteria are: M. kansasii, M. xenopi, M. simiae and especially mycobacteria belonging to the complex: M. avium-intracellulare (MAI complex). However, geographical variations exist in the incidence of the infection by these various opportunistic mycobacteria.
In a recent study carried out by the Laboratory of Bacteriology and Virology of the Pitie-Salpetriere hospital group directed by Professor J. Grosset, it was shown that among the 709 AIDS patients whose blood was cultured (2460 samples) 63 (8.9%) had positive haemocultures with mycobacteria. On the other hand, 84.1% of the positive haemocultures contained M. avium-intracellulare and 11.1% M. tuberculosis.
M. avium and M. intracellulare are two phenotypically related species indiscernible by conventional identification, and are therefore grouped into an MAI complex. MAI complex for the purposes of the present invention is understood to mean the strains as defined by SAITO et al. (J. Clin. Microbiol., August 1990, 28, p. 1694-1697). This complex brings together human and animal pathogens. In man non-infected by the HIV viruses, the infections are especially located in the lungs in adults and in the ganglia in children. MAI infections are diagnosed in 50 to 90% of AIDS patients. The locations are, in this case, preferably in the digestive system and are disseminated. MAI is also a pathogen which is important in veterinary medicine, affecting poultry, bovine, caprine and ovine species and the like. The animal pathogens, M. paratuberculosis and M. silvaticum, were recently introduced into the nomenclature as sub-species of M. avium (Thorel et al., Int. J. Syst. Bacteriol., 1990, 40, 254-260). It should also be noted that strains of M. paratuberculosis were isolated from patients suffering from Crohn's disease.
Using serology, 28 serotypes are recognised within MAI. Currently, serotypes 1 to 6, 8 to 11 and 21 are classified as M. avium and serotypes 7, 12 to 20 and 25 as M. intracellulare. An uncertainty remains as to the taxonomic position of serotypes 22 to 24 and 26 to 28. In man non-infected with the HIV viruses, the distribution of the serotypes is highly dispersed whereas in AIDS patients, only serotypes 1, 4, 8 and 9 are predominant (data of the National Centre of Reference for Mycobacteria). This distribution is similar to that observed in animals where serotypes 1, 2, 3, 4, 8 are preferably found. The French epidemiological situation for the M. avium strains isolated from AIDS patients is different from that observed in the United States or in Sweden. While in France serotype 8 is predominant, serotypes 4 or 6 are the ones found in these other two countries.
The serotypes (or serovars) mentioned in the present patent are as defined by P. J. Brennan et al. (Identification of A typical Mycobacteria by Thin-Layer Chromatography of Their Surface Antigens, Journal of Clinical Microbiology, Oct. 1978, p. 374-379.
Opportunistic mycobacteria pose numerous challenges to the microbiologist, the immunologist and the clinician. Indeed, unlike M. tuberculosis which has a homogeneous bacterial population, the strains of the M. avium-intracellulare complex are composed of cells which give various types of colonies on culture; a discrepancy was observed between the response of patients to antituberculous chemotherapy and the results of the antibiogram in vitro, calling into question the role of antibiograms (in any case, the MAI infections respond barely or poorly to various antibiotic and antitubercolous treatments or the like); mycobacteria are responsible for the non-specific sensitization to tuberculin and can also affect the immune response.
In the laboratory, the difficulty consists in isolating opportunistic mycobacteria from various samples and then in determining the species. The ubiquitous presence of opportunistic mycobacteria in the environment often renders the distinction between colonization and infection difficult. That is why it is important to refer to precise clinical and bacteriological criteria before deciding on the clinical significance of a mycobacterinm isolated from a sample. Consequently, it is essential to establish a cooperation of good quality between clinicians and bacteriologists and to have reliable and specific techniques for identifying opportunistic mycobacteria. It is evident that the mycobacterial species isolated and identified from pathological products are linked to the isolation techniques and to the schemes used nowadays for their identification. Bacteriologists know that the identification of opportunistic mycobacteria is made difficult by several elements: certain species are more or less sensitive to the decontamination techniques required before the culture, several species are isolated in primary culture only under certain conditions, finally, certain identification techniques based on phenotypic properties are not sufficiently reliable and can lead to a wrong identification. On the other hand, the taxonomic status of certain mycobacteria related to the MAI complex still remains uncertain and, in this case, the identification cannot be established.
Mycobacteria of the M. avium-intracelllulare complex can, like M. tuberculosis, be responsible for adenites in children. The clinician frequently gives an initial antituberculous treatment while awaiting the microbiological diagnosis which may take up to several weeks, this treatment being stopped as soon as the microbiological diagnosis is obtained.
In order to avoid these errors of interpretation and to permit a rapid diagnosis, the inventors responsible for the present invention set themselves the objective of isolating DNA sequences specific for mycobacteria belonging to the M. avium-intracellulare complex and of developing, using these sequences, a method of identification based on the PCR technique and the molecular hybridization technique. They therefore searched for sequences which can be used both for the diagnosis and for the molecular epidemiology.
Recently, approaches using molecular hybridization to identify M. avium-intracellulare species have been proposed; however, most of these methods permit identification only after culture since they are not sufficiently sensitive to detect these mycobacteria directly in the biological samples. This is the case for example for the cDNA probe which is complementary to ribosomal RNA sequences and marketed by Gene-Probe (San Diego, Calif.), see WO 84/02721, WO 88/03957, Musial, C. E. et al. (J. Clin. Microbiol., 1988, 26, 2120-2123). Furthermore, mycobacterial strains according to the criteria of Saito et al. (J. Clin. Microbiol, 1990, 28, 1694-1697) are not detectable using the probes marketed by Gene-Probe. Other authors have found similar results: Enns, R. K. (Lab. Med. 1988, 19, 295-300), Sherman, I. et al. (J. Clin. Microbiol., 1989, 27, 241-244).
FR-2,645,878 describes, in addition, a nucleic sequence of 383 base pairs homologous in at least 5 mycobacteria species including M. tuberculosis.
This sequence has, however, the disadvantage of not being specific for the M. avium-intracellulare complex.
A more sensitive and more specific method was therefore necessary in order to establish a reliable diagnosis for atypical mycobacterioses and a precise identification of the various opportunistic mycobacteria.